Aquatic photosynthetic organisms including Chlamydomonas reinhardtii, can adapt to the low-CO₂ stress conditions by inducing the carbon-concentrating mechanism (CCM). During this acclimation process, the expression of Cah1 encoding a periplasmic carbonic anhydrase is induced by CCM1 and LCR1, which is MYB-type transcription factor, under low-CO₂ condition in light. Cah4, encoding a mitochondrial carbonic anhydrase, is also induced under the light and low-CO₂ condition. Both Cah1 and Cah4 are suppressed under high-CO₂ condition, but regulator(s) involved in this suppression is unknown. To identify novel regulatory factor(s) involved in the expression of Cah4, the luciferase gene (lucNC) was joined to the 2-kb upstream region of Cah4 (Cah4p::lucNC) and introduced into wild type cells. One of the transformants showed 35 times higher luciferase activity under low-CO₂ condition than under high-CO₂ condition. Subsequently, the hygromycin-resistant gene cassette was introduced into this Cah4p::lucNC strain, and transformants that show reduced or increased luciferase activity under low- or high-CO₂ conditions, respectively, were screened for.

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This study aims to construct plasmid vectors useful to measure promoter strengths in cyanobacterium Synechococcus elongatus PCC 7942 and to analyze quantitatively various DNA sequences. Luciferase gene (luc) from firefly (Photinus pyralis) as a reporter was obtained as SD-containing luc gene from pSP-luc+NF (Promega) and cloned within a linker inserted between SacI and XhoI sites in pUC303, resulting in a promoter-probe vector, pLUC1. Various blunt-ended DNA fragments were cloned into SrfI site upstream the luc gene on pLUC1, which were used to transform E. coli MOSblue as well as streptomycin-resistant (rps12-R43) GRPS1 strain derived from S. elongatus PCC 7942 R2-SPc.
The psbAI promoters (193bp, 97bp, 55bp) from PCC 7942 and lacZ promoter (107bp) from E. coli were cloned into pLUC1, resulting in pLUC1-psbAI-193, -97, -55 and pLUC1-lacZ-107, respectively. Specific activities of luciferase in transformants of GRPS1 carrying recombinant plasmids allowed us to confine psbAI promoter regions that might interact with positive or negative regulatory factor(s).

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A polyubiquitin gene (McUBI1) promoter region including its 5' UTR together with its first intron was isolated from the genomic DNA of M. crystallinum using a primer walking protocol. A 726 bp intron was found immediately 5' to the translation initiation codon in the second exon. To establish this McUBI1 promoter as a native and constitutive promoter, we have improved a transient expression assay based on particle bombardment and the dual-luciferase reporter technique, which should facilitate investigations into the regulation of McUBI1 gene in vivo. An intronless 2.58 kb promoter fragment was fused with the firefly luciferase and Renilla luciferase reporter gene. These chimeric gene constructs were introduced into leaves of M. crystallinum by particle bombardment for transient assay. We observed high expression level from the intronless promoter in M. crystallinum which is more stable than that from CaMV35S promoter. We will investigate the effect of existence of intron on expression level in M. crystallinum.

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A gene encoding carotenoid cleavage dioxygenase (CmCCD4a) is specifically expressed in white chrysanthemum (Chrysanthemum morifolium Ramat.) petals and brings about white coloration via the cleavage of carotenoids (Ohmiya et al., 2006). In order to obtain a stable promoter that induces exogenous gene expression specifically in the petals, we isolated and characterized the CmCCD4a promoter. We obtained approximately 3 kb upstream of CmCCD4a by using the TAIL-PCR method. A transient expression assay performed using the particle bombardment transformation system revealed that the 1.2-kb region sufficiently mimicked the promoter activity and that 2 cis-regions, designated cis1 and cis2, were involved in ray petal-specific expression. In addition, we found that chimeric promoters containing a combination of cis1/2 and the 1.2-kb CmCCD4a promoter enhanced both promoter activity and the organ specificity of the original CmCCD4a promoter. We are now growing transformed chrysanthemums harboring chimeric CmCCD4a promoter constructs fused to the beta-glucuronidase (GUS) reporter gene.

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Squalene synthase (SS) synthesizes the first lipophilic precursor squalene in phytosterol synthesis pathway (Suzuki et al 2002, Akamine et al 2003, Lee et al 2004, Uchida et al 2009). In a microalga Botryococcus braunii, methylsqualenes and their derivatives are released to extracellular matrix as secondary metabolites. The cDNAs clone of B. braunii SS gene (BbSS) was isolated and functionally characterized (Okada et al 2000). Prominent SS enzyme activity was detected transiently immediately after inoculation into a fresh medium, and weaker activity was observed continuously in the other culture periods. In order to analyze gene expression of BbSS, we have isolated corresponding genomic clones through PCR amplification and cloning to obtain the 0.55-kb and 1.4-kb clones. Nucleotide sequencing of these two clones revealed existence of 223-bp and 756-bp introns after 2nd base of 52nd amino-acid residue and 3rd base of 232nd amino-acid residue, respectively. We are further isolating other genomic clones spanning in the entire regions of the gene, including the 5' upstream region.

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Chlamydomonas reinhardtii is a useful organism to analyze unidentified gene functions because of its haploid nucleus. Into the genome of a Chlamydomonas strain 19-P(1030) that possesses an aadA transgene conferring spectinomycin resistance, we introduced a aadA mRNA-targeting silencer-construct, which consists of an inverted repeat of the aadA ORF and a ble marker gene to obtain a transformant (RNAi-37). In this transformant aadA mRNA was reduced to about 20% of the 19-P(1030). A tag-aphVIII DNA was introduced into the RNAi-37 to obtained a transformant 92-12C, in which RNAi efficiency was apparently reduced and a 14-kb genome region was deleted along with the tag insertion. This genome region contains three putative ORFs encoding an unidentified protein, a thioesterase-like protein and a protein with PWI/zinc-finger motives. We have obtained three BAC-subclones containing each of the three genes with promoter region and one for the entire deletion region, and are transforming the 92-12C with the plasmids to identify the gene that is involved in the reduced RNAi efficiency.

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RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. RISCs invariably contain ARGONAUTE (AGO) family proteins and single-stranded small RNAs. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the in vivo process. We demonstrate that a cleavage-competent RISC can be assembled with a synthetic small interfering RNA (siRNA) duplex in the extracts of evacuolated tobacco BY-2 protoplasts (BYL). In the BYL system, the siRNA duplex was incorporated into AGO1 by a mechanism requiring ATP, Mg²⁺ and the molecular chaperone HSP90, and the removal of passenger strand of siRNA duplex depended on the RNase activity of AGO1. Directed by the guide strand siRNA sequestered to AGO1, the assembled RISC bound and cleaved the complementary target RNA.

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The moss Physcomitrella patens is an excellent model plant in the reverse genetics because high efficiency-gene targeting is applicable to this moss. In this study, we have developed a gene silencing system using artificial micro RNA (amiRNA). Micro RNAs are approximately 21-nt. The secondary structures of miR319 precursor transcripts from Arabidopsis and Physcomitrella are highly conserved. AmiRNA from Arabidopsis miR319a has been successful in the suppression of gene expression (Schwab et al. 2006). To develop a gene silencing system using amiRNA, we constructed the stable moss transgenic line that constitutively expressed a nucleus localized GFP fusion protein. Then, we designed an amiRNA to target the GFP gene and constructed a plasmid consisting of the amiRNA precursor sequence and actin or CaMV35S promoter. The amiRNA constructs were introduced into the moss by particle bombardment and we examined whether the expression of GFP in the transgenic line was transiently suppressed. These results will be presented.

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NMD (Nonsense-mediated mRNA Decay) mRNA surveillance conserved among eukaryotes is involved in degradation of aberrant mRNAs that contain premature termination codon (PTC) and in the translation-dependent processing of mRNAs with various structural features. Knockout of AtUPF1 or AtUPF2 encoding major factors of NMD in Arabidopsis are lethal and lba1 missense mutant of AtUPF1 shows pleiotropic phenotypes. mRNAs that increased in lba1 and atupf3-1 compared to the wild type were enriched with PTC⁺-mRNAs produced by alternative splicing and mRNAs with short ORF in the 5'-untranslated region. Among them, alternatively spliced PTC⁺-mRNA of TFIIIA and uORF-containing mRNA of SAC51 encoding a bHLH protein seem to be direct targets of NMD, since they showed longer half-lives in lba1 and atupf3-1 compared to the wild type and their degradation was translation-dependent. Seeds of lba1 and atupf3-1 were larger at long axis than the wild-type seeds, which was due to the longer distance between the ovule primordia formed on septum during pistil development. We are searching for mRNAs that are targets of NMD and involved in the determination of periodical pattern of ovule primordia formation.

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Since transposons are potentially highly mutagenic, the majority of them are epigenetically silenced by RNA silencing machinary. However, most proportion of the eukaryotic genome is composed of transposoable elements. This indicates that transposons have likely evolved countermeasures to transposon silencing.
Here we show that a class of transposons in rice utilizes a microRNA (miRNA) as a strategy to suppress silencing. We found that miR820 family is a miRNAs, which are expressed from CACTA DNA transposons in rice and whose target is a DNA methyltransferase gene, OsDRM1a, one of the components of epigenetic silencing. First, we tested if miR820 regulates the expression of OsDRM1a by using small RNA deficient mutant in rice. In this mutant, the expression level of miR820 and OsDRM1a reduced and increased, respectively. This result indicates that miR820 negatively regulates the expression of OsDRM1a. Next, we suppressed the expression of OsDRM1a by RNA interference. This resulted in increase of transposon encoded gene expression. Taken together, our results demonstrate that miR820 suppresses transposon silencing by down-regulation of OsDRM1a expression.

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The acl5 mutant of Arabidopsis thaliana shows a severe defect in stem elongation. ACL5 encodes thermospermine synthase. Thermospermine is a structural isomer of spermine. sac51-d was identified as a suppressor mutant of the dwarf phenotype of acl5 and its responsible gene SAC51 encodes a bHLH transcription factor. sac51-d contains a point mutation in the 4th of five uORFs present in the 5' leader sequence of SAC51 mRNA and increases translation of the main ORF, which may cause the recovery of stem growth. We have generated transgenic lines carrying the GUS gene fused with the SAC51 promoter and its 5' leader, in which each one of the uORFs is disrupted. Only the plants with the 4th uORF-disrupted construct showed higher levels of the GUS fusion transcript and the GUS activity than those with the wild-type construct but less increase by external therspermine than those with the other constructs tested. The SAC51 promoter-GUS construct lacking most of the SAC51 5' leader was not responsive to thermospermine. These results suggest that thermospermine acts in overcoming the 4th uORF-dependent repression of the main ORF translation and/or regulating the mRNA stability of SAC51.

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The 20 amino acids, except for methionine and tryptophan, are coded for by 2 to 6 codons called synonymous codons. Synonymous codons are not used with equal frequency in protein coding sequences, and are used differently by different organisms. Although it has been thought that the codon usage is correlated with the translation efficiency, we found that translation efficiencies of synonymous codon groups are not always correlated with their usages in tobacco chloroplasts. This finding suggests that the inefficient codons in translation are selectively used in the several chloroplast mRNAs. Thus, we calculated the codon frequencies of individual chloroplast mRNAs, and found that tobacco rps16 mRNA contains many inefficient codons. Here, we analyzed the effect of inefficient codons in rps16 mRNA on its translation efficiency.

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COP9 signalosome (CSN) is a nuclear complex regulating signal transduction in plants and animals. To dissect the function of CSN, we focus on the function of CSN1 subunit. The N-terminal region of CSN1 (CSN1N) possessed a transcriptional repression activity in mammals, and was essential for survival in plants. To understand this mechanism, Yeast Two-Hybrid method was utilized to isolate proteins interacting with CSN1N in Arabidopsis. Here, we isolated a transcription factor containing a predicted structural domain composed of three helixes and two loops, a conserved signature domain of the Trihelix protein family in Arabidopsis. Through a series of pull-down assays, this transcription factor was shown to directly bind CSN1N in a specific manner. Here, we report the gene expression pattern of this transcription factor and the phenotype of its mutants. We will further characterize this transcriptional factor, and discuss its role in CSN mediated signal regulation.

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Ubiquitin(Ub)/26S proteasome pathway plays an essential housekeeping role to eliminate the proteins which are damaged or misfolded. It is also essential for aspects of cellular regulation by removing short-lived regulatory proteins as a way to fine-tune homeostasis, adapt to new environments, and redirect growth and development. The 26S proteasome consists of two multisubunit complexes, 20S core particle (CP) and 19S regulatory particle (RP). The RP contains thirteen non-ATPase subunits (RPN) and a ring of six AAA-ATPase subunits (RPT).
We reported RPT2a and RPT5a deficient mutants resulted in enlarged leaves, which are caused by accelerated endoreduplication.
To elucidate the function of AtRPT2a, we explored interacting factors with AtRPT2a by proteome analysis. Here we report the functional analyses of the candidates.

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Arabidopsis thaliana BOR1 is a B transporter required for xylem loading of B under B limitation. It has been already reported that BOR1 is selectively degraded under high B supply. Here we show that BOR1 5'UTR is also involved in regulation of BOR1 expression/accumulation in response to B availability.
BOR1 promoter containing 3 kb genomic sequence upstream from the translational start site was fused to GUS and introduced to A. thaliana. Relative values of GUS activities to mRNA levels of GUS under the high B concentration (3 mM) were decreased to ca. 40 % of those under the low B condition (0.3 μM) in roots. When 5'UTR was removed from the promoter fragment, no significant difference was found in the relative values of GUS activities to mRNA levels between the two B conditions. The relative values under the low B treatment were not statistically different between the two constructs. These results suggest that BOR1 translation is repressed under high B supply and 5'UTR mediates this B-dependent repression.

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Glucosinolates are a class of secondary metabolites produced by Brassicaceae plants, serving as antiherbivore compounds. Glucosinolates are classified according to the use of different amino acids as the biosynthetic precursors: aliphatic glucosinolates are derived from methionine, and indolic glucosinolates are produced from tryptophan. It has been reported that a variety of MYB-like transcription factors are involved in the regulation of glucosinolate biosyntheses. MYB38/ATR1, MYB51, and MYB122 are involved in the regulation of indole glucosinolate biosyntheses, while MYB28, MYB29, and MYB76 are implicated in the aliphatic glucosinolate biosyntheses.
Here, we report the identification of a novel transcription factor involved in the gene expression network regulated by these previously characterized MYB transcription factors. We discuss possible regulation mechanisms of the glucosinolate biosyntheses involving this newly identified transctiption factor in terms of glucosinolate profiling analyses and gene expression profiling studies.

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The biosynthesis of camalexin, a major phytoalexin in Arabidopsis, is induced upon infection of a variety of pathogens, and abiotic stresses such as AgNO₃ treatment and UV-B irradiation stimulate the biosynthesis as well. Recent studies have almost elucidated the entire pathway of camalexin biosynthesis pathway including several cytochromes P450. The expression levels of such biosynthetic genes have been demonstrated to be greatly enhanced under those inducing conditions. However, possible mechanisms underlying the induction of camalexin biosynthesis remain elusive.
Here we report identification and characterization of transcriotion factors involved in the induction of camalexin biosynthesis. One of T-DNA insertion events within transcrioption factor genes resulted in a significant reduction of the camalexin accumulation levels. We discuss possible regulation mechanisms of the camalexin biosynthesis involving the induction of this transctiption factor gene in terms of transcriptome analyses.

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In Arabidopsis, flavonoid biosynthesis is regulated by MYB transcription factors such as MYB75/PAP1 for anthocyanin and MYB12 for flavonol. We have generated the transgenic lines over-expressing both PAP1 and MYB12 (WOX1-1,2) (1) to characterize them besides the lines of each single MYB gene over-expression (pap1-D and MYB12OX) by metabolome and transcriptome analysis. The flavonoid profiling exhibited that the amounts of flavonoids in WOX1 were the additive levels of anthocyanins and flavonols due to the over-expression of PAP1 and MYB12. Transcriptome analysis showed that 81 genes were specifically up-regulated in WOX1. The gene ontology analysis indicated that 57 genes, which are categorized into 'response to stimulus', were up-regulated in WOX1. Because most of them are involved in the stress response mechanism, stress experiments (salt and methyl viologen) were conducted. These experiments showed that pap1-D and WOX1 mutants exhibit the tolerance against those stresses. These results suggested the feed-forward interaction from flavonoid biosynthetic mechanism to stress mechanism.

Reference
(1) Nakabayashi R, et al., (2009) Phytochemistry, 70: 1017-1029

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Whereas higher plants commonly produce phenylpropanoids, limited plant species produce alkaloids. Among alkaloids isoquinoline alkaloids are a major group and its biosynthetic pathway has been intensively investigated at the molecular level. Especially, berberine biosynthesis in Coptis japonica (Ranunculaceae) has been well characterized and a general transcriptional factor, i.e. CjWRKY1 was isolated (Kato et al. , 2007). Here, we report the isolation and characterization of another general transcriptional factor, CjbHLH1. CjbHLH1 was isolated using transient RNAi analysis as CjWRKY1. However, the transient overexpression of CjbHLH1 decreased the expression of berberine biosynthetic genes. To characterize the functionality of CjbHLH1, we analyzed subcellular localization of CjbHLH1 using GFP fusion construct and its transcriptional activity with biosynthetic gene promoter::luciferase reporter gene (pLUC). Transient expression of CjbHLH1:GFP in Cj protoplasts confirmed nuclear localization of CjbHLH1. Further transient assay using pLUC reporter indicates the transcriptional activity of CjbHLH1. Isolation of CjbHLH1-interacting factors is on going.

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Plants produce not only oxygen and food, which are essential items for animals to survive, but also various metabolites such as medical materials. Manipulation of a metabolic pathway by genetic engineering would be a useful strategy to alter useful metabolites quantitatively and/or qualitatively. Because it becomes evident that transcription factors organize the expression of multiple genes for metabolic enzymes that act in the same metabolic pathway, it is expected that modification of a transcription factor would be efficient to control of metabolic pathway and to enhance production of useful compounds. We developed a novel gene silencing system, called Chimeric Repressor Gene- Silencing Technology (CRES-T), in which a transcription factor that is converted into strong repressor by fusion with EAR-motif repression domain (SRDX), induce dominant negative phenotype similar to loss-of-function of the alleles of the gene for transcription factor by suppressing target genes dominantly. Using CRES-T system, we are screening transcription factors that could regulate metabolic pathway. We successfully identified several transcription factors that regulate various metabolic pathways.

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The petals of cypress vine (Ipomoea quamoclit) may be red, pink, or white; this difference in colors is due to the presence of varying amounts of pelargonidin-based anthocyanin. In this study, we aimed to elucidate the mechanism that controls anthocyanin accumulation in I. quamoclit petals at the molecular level. Pelargonidin was not detected in unhydrolyzed extracts of petals of the white-flowered I. quamoclit but was detected in hydrolyzed extracts; this indicates that leucopelargonidin, a precursor of pelargonidin, is synthesized in the petals of the white-flowered I. quamoclit. In petals of the white-flowered I. quamoclit, anthocyanidin synthase (ANS) expression was not detected, whereas dihydroflavonol 4-reductase (DFR) and flavanone 3-hydroxylase (F3H) expression was detected. Moreover, all progenies obtained by interspecies crossing between white-flowered I. quamoclit and yellow-flowered I. hederifolia, which lacks DFR expression, contained pelargonidin in their petals. These results suggest that pelargonidin was produced in the petals of the progenies by complementary expression of each gene.

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Flower color is one of the most important factors affecting the commercial value of ornamental plants. Recently, green-hued flowers have become popular for commercial purposes. However, the pigment composition of green flowers and their mechanisms of color development are totally unknown. We analyzed the pigment composition of various green flowers by HPLC. We revealed that some green flowers, for instance, lisianthus (Eustoma grandiflorum), contain chlorophyll and pheophytin a. Pheophytin a is generated by removal of the central magnesium atom from the porphyrin macrocycle of chlorophyll a. Pheophytinase (PPH) was recently found to catalyze the dephytylation of pheophytin a in the chlorophyll-degradation pathway (Schelbet et al., 2009, Morita et al., 2009). The leaves of pph mutants are reported to accumulate pheophytin a and exhibit the "stay-green" phenotype. We therefore presumed that PPH may be involved in the green coloration of the petals. Next, we isolated the PPH gene from E. grandiflorum and examined its expression levels in green and white flowers.

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Caffeine and theobromine are purine alkaloids that are present in high concentrations in plants of some species of Camellia. The main caffeine biosynthetic pathway involves in three S-Adenosyl-_L-methionine (SAM)-dependent methylation steps. SAM-dependent N-methyltransferases play an important role in the regulation of caffeine biosynthesis. Caffeine synthase catalyzes 1-N and 3-N-methylation reaction of mono- or di-methylxanthines, on the other hand, theobromine synthase catalyzes 3-N-methylation reaction of 7-methylxanthine. Recently we reported occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.
In this study, we isolated caffeine synthase orthologous genes from 12 species of Theaceae. Three recombinant enzymes which were approximately 70% homologous to tea caffeine synthase did not show theobromine synthase activity. The molecular evolutions of these methyltransferases were discussed.

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Some furofuran lignans have beneficial effects on maintaining health. Our aim is to produce furofuran lignans efficiently by metabolic engineering of plants. In this study, we conducted lignan profiling in metabolic engineered Arabidopsis to survey the effects of modification of the biosynthetic pathways on lignan production. CYP81Q1 from sesame was introduced in Arabidopsis, resulting in newly formation of a furofulan lignan. Additionally, the amount of the furofulan lignan was increased by knockdown of a pinoresinol/lariciresinol reductase (PLR). We concluded that it is possible to produce furofuran lignans by metabolic engineering of plants.

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We present the engineering of lignan biosynthesis in Forsythia koreana cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable to that obtained from the leaves. To increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of a pinoresinol/lariciresinol reductase, PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing PLR-RNAi and CYP81Q1, which is a cytochrome P450 enzyme responsible for the biosynthesis of sesamin from pinoresinol, exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production.

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In this study, we attempt to engineer the prenylation reaction in the major fruits crop tomato (Solanum lycoperisicum L). Tomato plants were transformed with prenyltransferase cDNAs encoding SCO7190 (HypSc) and SfN8DT-1, which are derived from a Streptomyces strain and Sophora flavescens, respectively. These enzymes can specifically catalyze the addition of a dimethylallyl group to naringenin at the C6 and C8 atom, respectively, under the control of the fruit-specific E8 promoter. LC-MS analysis demonstrated that the presence of 8-dimethylallylnaringenin in tomato fruits overexpressing SfN8DT-1, whereas unexpectedly fruits overexpressing of SCO7190 (HypSc) produced 3-prenylnaringenin unlike in vitro assay using the recombinant protein. On the other hands, the Arabidopsis plants overexpressing SfN8DT-1 accumulated 8-dimethylallylkaempferol, which is also unlike the in vitro data of this prenyltransferase. Our results suggested that the product specificities could be largely dependent on the host plant species.

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Sesquiterpene lactone (STL) is a characteristic secondary metabolite most often found in Asteraceae family. Despite the pharmaceutical importance of STLs, most of their biosynthesis remains unknown at the molecular level. Since costunolide is the simplest type of STL and is believed to be a central precursor of diverse STLs, we have focused on the understanding of costunolide biosynthesis as an important first step for STL biochemistry.
Costunolide is biosynthesized from farnesyl diphosphate (FPP) via germacrene A and germacrene A acid (GAA). While FPP is converted to germacrene A by a terpene synthase, the gene of which is already known, we recently identified the gene of germacrene A oxidase that catalyzes 3 step-oxidation from germacrene A to GAA. Since the last step from GAA to costunolide is reported as a P450 reaction, we are trying to isolate the corresponding P450 gene using sunflower trichome EST library and lettuce EST information. Although the difficulty was to obtain GAA, the substrate of the targeted P450, we overcame it with the aid of synthetic biological approach; i.e., we expressed GAA biosynthetic genes in engineered FPP high-producing yeast to produce GAA.

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Alkaloids play a key role in the plants' defense mechanism against pathogens and herbivores. The major alkaloid of the Nicotiana species, nicotine, is translocated via xylem transport from the root tissues where it is biosynthesized to the accumulation sites, the vacuoles of leaves. However, its transport mechanism is still largely unknown. Recently, transcriptome analysis of methyl jasmonate-elicited tobacco BY-2 cells identified four transporter genes (Nt-JAT1, C215, T408, T449) which were highly up-regulated in a coordinate manner with the nicotine biosynthetic genes. Furthermore, Nt-JAT1, MATE transporter, was suggested to be involved in the vacuolar accumulation of nicotine in tobacco leaves.1) In this study, we characterized Nt-T408, which belongs to Nucleobase cation symporter-1 (NCS1) family protein. Hydrophobicity profiles of Nt-T408 suggested the presence of 12 transmembrane domains. Nt-T408 was expressed in roots, stems, and leaves, and localized in the tonoplast of leaf cells. Its functional analyses concerning the nicotine transport using heterologous host organisms, such as yeast and insect cells will be also presented.
1) Morita et al. (2009) PNAS 106: 2447-2452.

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Boron (B) is essential for plant growth but toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel NIP5;1 and a boric acid/borate exporter BOR1 are required for efficient B uptake and subsequent translocation into xylem, respectively. However, under high B conditions, BOR1 activity is repressed through endocytic degradation presumably for avoiding B toxicity. When expressed under control of the native promoter, GFP-NIP5;1 was localized preferentially in outer (distal) plasma membrane (PM) domains in root epidermal cells. In contrast, BOR1-GFP was localized preferentially in outer (distal) plasma membrane (PM) domains in various root cells including epidermal and endodermal cells. In this presentation, we would like to discuss about the mechanisms of the polarized localization of these transport proteins based on following observations. 1, Estimation of lateral diffusion rates of GFP-NIP5;1 and BOR1-GFP in the PM by FRAP analysis. 2, Detailed analysis of GFP-NIP5;1 and BOR1-GFP localization in PM and endosomes.

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It has been well characterized that glycerol 3-phosphate (G3P) is exchanged with orthophosphate by the G3P permease (G3PP) in the inner membrane of Escherichia coli. Although no eukaryotic G3PPs have been identified so far, genes homologous to the bacterial G3PP are conserved among eukaryotes including higher plant. Arabidopsis has five G3PP homologs (AtG3PP1 to AtG3PP5), and according to the microarray database GENEVESTIGATOR, the expression of AtG3PP3 and AtG3PP4 are increased by salt, osmotic and cold stress. We reported in the T-DNA-insertional mutant g3pp4, germination of seeds was disturbed more severely than wild type under salt stress(Sakuma et al., 2008).
In this study, we further analyzed g3pp4 mutant together with g3pp3. Under osmotic stress with 300 mM mannitol, as well as under salt stress, g3pp4 mutant showed delayed and decreased germination. In addition, weight of g3pp4 mutant seeds was reduced by 21%, suggesting reduced oil content in seeds. In contrast, no clear difference was found between g3pp3 mutant and wild type. We will further investigate the involvement of AtG3PP4 in stress tolerance and lipid metabolism.

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We previously detected that plant acetylcholinesterase (AChE) was in the cortex of coleoptile node of maize seedling by histochemical and DTNB method. We also identified AChE genes from maize and siratro seedlings. In this report, subcellular localization of the plant AChEs (maize AChE, rice AChE1 and rice AChE2) in leaf tissues of the transgenic rice plants was investigated. Two rice AChE genes were identified high AChE homologous genes based on our cloned maize AChE gene. Then, maize AChE and two rice AChEs were introduced rice plants, respectively. Localization of these AChEs was investigated by immunofluorescence in leaf tissues using antibody against maize AChE. The maize AChE and two rice AChEs were localized in extracellular spaces in the leaf of the plants. The results suggest that acetylcholine (ACh), ACh receptor (AChR) and AChE (ACh-mediated system) may function at extracellular region. Now we continued investigate localization of green fluorescent protein (GFP) fusion AChE in transgenic rice. This research was supported by grant from Japan Space Forum.

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Agar is substantially contaminated with several elements including non- and essential plant mineral nutrients (Jain et al. Plant Physiol.150.1033). This makes it difficult to analyze nutrient deficiency using medium solidified with agar. Hydroponic cultures are not suitable for handling a large number of plants. In this study, we developed an experimental system that can avoid contamination and is suitable for handling a large number of plants. We used urethane foam for growing Arabidopsis and checked if this method is applicable for nutrient studies.
For each element contained in our standard nutrient solution, we prepared nutrient solution without it and observed the symptoms. Except for Co and Cl, we were able to see clear deficient symptoms. Growth properties were different between Col-0 and Ler under Mo and B deficient conditions. We are currently analyzing the Zn and Mg deficiency in detail and screening for nutrient deficiency-sensitive mutants.

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We present the identification study of phosphate transporter genes in Lotus japonicus. mRNA expression of phosphate transpoters in tissues was analyzed by real-time PCR and in situ hybridization. It was found that, these six phosphate transpoters were expressed in root, leaf, stem, flower and pod with different patterns. Under phosphate starvation condition, expression level was increased in root, leaf, and stem. On the other hand, expression of flower, and pod was the same level as that in control. Especially, in case of LjPT1, LjPT2 and LjPT3 that increased considerably under phosphate starvation in root and leaves.
The expression of LjPT1 was increased earlier than these of the other genes in root, whereas LjPT1 and LjPT2 expressions ware increased in matured leaf. The expression of all phosphate transporters was increased in young growing leaf. Then, the amount of phosphate translocation to these tissues was determined by successive ³²P images obtained by real-time imaging system we developed. The amount of phosphate translocation per hour was 5 fold higher in phosphate deficient samples than that in control sample.

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We studied absorption and translocation manner of Mg using Oryza Sativa L. cv. Nipponbare and suggested the involvement of transport proteins in our previous report. In this study, the role of nine proteins in total were studied (OsMGTs). In the case of amino-acid sequence of OsMGTs, these candidates belonged to 2-TM-GxN type transporters, found in the CorA super-family proteins that distributed widely in nature. The ORF region of OsMGT was inserted to the plasmid, and was introduced to the yeast mutant lacking Mg transport activity. Then the yeast was grown on a plate medium or in culture solution containing different concentration of Mg. It was found that among the yeast tested, three OsMGTs showed the functional complementarity of Mg transport. There is a possibility that each OsMGT has different role(s) for Mg transport and homeostasis. It was reported that some transporters of CorA super-family have different cation selectivity. Therefore, the transport capability of other cations such as Co²⁺ is now under analysis.

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Vegetable crops grown on a high nickel-contaminated soil often show toxic symptoms, due to the over-accumulation of nickel. However, we have limited information on the molecular mechanism of nickel uptake, and this prevents to create an effective solution. This time we proposed a hypothesis that excess nickel was miss-transported by the transporters of other heavy metals, and examined the possibility of excess nickel uptake via uptake system of iron, which is a primary micro essential nutrient, in root of Arabidopsis thaliana. Plant samples were cultured hydroponically under two iron condition (0 or 50 μM Fe(III)-EDTA), then exposed to 25 μM NiCl₂. As a result, iron-unsupplemented samples showed visible nickel phytotoxic symptoms and increased their nickel concentration 70% higher than those of iron-supplemented. Next we examined nickel transport activity of IRT1, a high-affinity iron transporter in root, using yeast-expression system. The strain expressing IRT1 was more sensitive to nickel, and showed 2-fold higher nickel accumulation compared to control. These results suggested that nickel is transported via IRT1 iron uptake system of root under excess nickel condition.

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We have identified both influx and efflux Si transporters from rice, barley and maize. They all show similar transport activity for Si in Xenopus oocytes. However, the Si uptake capacity greatly differs between rice and barley or maize. To understand the mechanism underlying the species-dependent difference in Si uptake, we compared the expression level and localization pattern of Si transporters between rice and barley. Analysis with absolute quantitative real time PCR showed that the mRNA level of Lsi1 and Lsi2 was several-fold higher in rice than in barley. When HvLsi1 or OsLsi1 were introduced into a rice lsi1 mutant under the control of HvLsi1 promoter, both HvLsi1 and OsLsi1 were localized at the distal side of root exodermis and endodermis, which is similar to the native rice Lsi1. Although the mRNA level in both transgenic lines was similar, Si uptake by HvLsi1-expressed rice was lower than in that OsLsi1-expressed lines. Our results suggest that both higher expression level and transport activity of Lsi1 contribute to a higher Si uptake capacity in rice. Similar analysis with Lsi2 is currently being undertaken.

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The SE-CC complex plays the central role in sucrose translocation through the phloem. During the sink-to-source transition of leaves, the SE-CC complexes in the minor veins acquire the sucrose translocation capacity, which includes the secondary plasmodesma formation in the cell wall between SE and CC. We previously isolated rsx1 mutants that showed restricted sucrose translocation from a source leaf to shoots and displayed nascent secondary plasmodesmata in the cell wall between SE and CC. RSX1 is hypothesized to digest pectate in the middle lamella of the cell wall and promote the secondary plasmodesmata formation. However, the exact function of RSX1 in this process and the roll of RSX1 in the construction of sucrose translocation pathways in source leaves remain to be elucidated. In this report, we created transgenic RSX1-sGFP rsx1-2 plants that successfully recovered from the defects in the sucrose translocation capacity. The subcellular localization of RSX1-sGFP in the leaf veins of the transgenic plants will be reported.

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Cadmium (Cd) is the toxic heavy metal for both plants and humans, and Cd accumulation in rice grains has become a major agricultural problem in Japan. In this study, we investigated the role of OsNRAMP1 in Cd uptake and transport in rice using two cultivars, Sasanishiki (japonica subspecies, low Cd in shoots) and Habataki (indica subspecies, high Cd in shoots).
An OsNRAMP1::GFP fusion protein localized to the plasma membrane in onion epidermal cells. The growth of the yeast expressing OsNRAMP1 was impaired in the presence of CdCl₂ compared to the yeast transformed with empty vector. Moreover, the Cd content of the OsNRAMP1-expressing yeast was higher than the vector control. The amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. The expression of OsNRAMP1 was detected in the roots and to a lesser extent in the shoots when grown without Cd. Under these conditions, the expression of OsNRAMP1 in the roots of Habataki was 5.8 times higher than that of Sasanishiki. The results suggest that OsNRAMP1 is involved in the cellular uptake and accumulation of Cd in rice.

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In acidic soils, aluminum (Al) hydrolyzed into Al³⁺ cations and it become a limiting factor for plant productivity. Al resistance is an important agronomic trait for worldwide crop production. Previous research reported that the toxic effect of Al can reduced by chelating Al with organic anions such as oxalate, citrate, or malate. In wheat (Triticum aestivum), malate exudation is the major organic anion involved with Al resistance, and Al-activated malate transporter, ALMT1, was identified and characterized. Although QTL analyses suggested that Al resistance in wheat is multigenic, physiological evidence for multiple mechanisms of Al resistance has not yet been documented, including activation factors of ALMT1. In this report, we selected 2 wheat lines which show same genotype and expression levels of ALMT1. Several genes were isolated with cDNA subtractive method using these 2 lines. Chromosomal location of these genes was investigated, and expression analyses of these genes were performed with several wheat lines that show different Al sensitivity.

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Most blue color in flowers is due to anthocyanin, and considerable proportion of blue coloration can be attributed to metal-complexed anthocyanins. Recently, we reported vacuolar localized iron-transporter in blue petal cells of Tulipa gesneriana. However the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. In Hydrangea macrophylla, Al³⁺ is involved in blue coloration and the anthocyanin is formed Al³⁺-complex in vacuoles. To identify the molecular mechanism of blue coloration in hydrangea flowers, we tried to isolate the related genes transporting that metal ion into vacuoles. In this study, we tried to isolate the genes related to transporting Al³⁺ into vacuoles in Hydrangea macrophylla. To isolate those genes, we used Al³⁺-sensitive yeasts introduced the Hydrangea macrophylla cDNA library. After several screening, Al³⁺ tolerant yeast strains were isolated. But no genes from Hydrangea macrophylla were included in those yeast stains. Now, we are trying other methods for isolating the Al³⁺ transporting related genes into vacuoles.

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Under phosphorus (P) deficient condition, plants secrete organic anions such as malate and citrate into the rhizosphere to mobilize insoluble P, which is bound with the metal ions such as iron and aluminum. It is considered that the organic acid transporters play very important roles on utilization of insoluble P in soils, although the P starvation responsive organic acid transporter genes have not yet isolated. This study aimed to isolate and analyze the organic acid transporters that responded to the P deficient condition.
RNA was extracted from Arabidopsis thaliana cultured with P deficient and P sufficient nutrient solution and used for the microarray analysis. Homologs for ALMT, MATE and ABC transporter were found as organic acid transporter candidates, which were upregulated by P starvation. Significant amount of mRNA accumulation in the P starved root was confirmed for all candidates by RT-PCR. Interestingly, the P starvation responsive ALMT has a natural antisense transcript, which might involve in regulation of the ALMT expression.

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Rice aquaporins form a large family consists of 33 members, suggesting significant and/or specific roles of the aquaporins on water and solute movement inside the plant body. In the present study, we report organ specific expression profiles and their time-dependent changes for 30 aquaporins in the aerial part of the rice seedlings from heading to maturing stage. Total RNA was successfully extracted from various organs including leaf, internode, node, panicle neck, panicle, anther, filament, pistil, lodicule and maturing seeds. Quantitative real-time PCR analysis for these aquaporin members enabled us to compare absolute copy numbers of aquaporin transcripts among the members and the organs. The results showed that specific aquaporin levels were extremely high in some organs such as maturing seeds, lodicules and anthers. In internodes, the expression profile was contrasting between the growing and the maturing zone. Diurnal changes in the aquaporin transcripts were different among leaves, internodes and nodes even between the same members. These results contribute to understand the role of the aquaporins in the shoots and reproductive organs, which have been largely unknown.

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It was shown that heterotrimeric G protein complexes may contain unknown subunits, in addition to α subunit, β subunit and γ subunit in rice (Plant J., 38, 320-331, 2004) and Arabidopsis (J. Biol. Chem., 283, 13913-13922). In order to purify complexes, we searched for the optimum detergents to solubilization of complexes containing the α subunit of rice heterotrimeric G protein. In the study, Nonidet P-40 (NP-40), Cholic acid, Brij 35 and Digitonin were compared for the solubilization of the complex from the plasma membrane. As results, the α subunit and the βγ dimmer was completely dissociated from complexes, when Digitonin was used for the solubilization of complexes. A large quantity of complexes containing the α subunit, β subunit and γ subunits were obtained in the case of NP-40, compared with these detergents. The crosslinking using disuccinimidyl suberate (DSS) was carried out for the stabilization of complexes. As results, the α subunit was crosslinked to the β subunit. Now we continue to purify the crosslinked complexes.

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A 23-amino acid peptide, AtPep1, and its homologues are endogenous amplifiers of innate immunity of Arabidopsis. AtPep peptides bind to AtPEPR1, a plasma membrane leucine-rich repeat (LRR) receptor kinase. Here we identify a plasma membrane LRR receptor kinase with 76 % amino acid similarity to AtPEPR1, designated AtPEPR2, and characterize its role in the perception of AtPep peptides and defense responses. Both AtPEPR1 and AtPEPR2 were transcriptionally induced by wounding, treatment with methyl jasmonate, AtPep peptides and PAMPs. The effects of AtPep1 application on defense-related gene induction and enhancement of resistance to Pseudomonas syringae pv. tomato DC3000 were partially reduced in single mutants of AtPEPR1 and AtPEPR2, and abolished completely in double mutants. Photoaffinity labeling and binding assays using transgenic tobacco cells expressing AtPEPR1 and AtPEPR2 clearly demonstrated that AtPEPR1 is a receptor for AtPep1-6, and that AtPEPR2 is a receptor for AtPep1 and AtPep2.

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Plants are often exposed to external conditions that adversely affect their growth, development and productivity. Almost cases of NaCl exposure induces the cell death thought the complex network such as cell volume changes, DNA damages, oxidative burst, inhibition of transcription and translation and mitochondrial depolarization. High concentration of NaCl also increases the organic osmolytes. However, high concentration of osmolytes also have toxic effects by operating almost similarly pathways of NaCl leading to cell death. In this study, we were focusing on hyperosmotic stresses (NaCl and sorbitol)-induced ROS generation and cytosolic Ca²⁺ increase as initial responses of plants, then the relations between these responses and signaling pathways (adaptation/cell death induction) were investigated. The involvement of peroxidase-mediated singlet oxygen (¹O₂) generation in hyperosmosis stress signaling was suggested. Addition, ¹O₂ generation induced Ca²⁺ signaling have the small role of adaptation of hyperosomosis changes.

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Histidine kinases (HKs) are involved in the perception of osmotic stress in cyanobacteria and yeast. We previously demonstrated that HKs AHK1 and AHK2/3 function as positive and negative regulators in osmotic stress responses in Arabidopsis, respectively, and suggested the involvement of HKs in the perception of osmotic stress. To clarify whether HKs-mediated osmotic stress responses are ubiquitous in higher plants, we focused on the roles of rice HKs in osmotic stress responses.
Through homology searches against the rice genome, we found four close homologs of cytokinin receptor genes (OsHK3, OsHK4, OsHK5 and OsHK6), but could not find any AHK1 homologs.Then, we cloned the ORFs of each OsHK gene and tested their functions in yeast cells. In complementation tests of yeast mutants, OsHK3, OsHK4, OsHK5 and OsHK6 could function as cytokinin receptors, and OsHK6 could function as an osmosensor in a cytokinin dependent manner.
Now we are analyzing the response of OsHK-overexpressing rice to cytokinin or osmotic stress treatments. The roles of these histidine kinases in rice will be discussed.

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We describe two Oryza sativa stress-related CCCH-type Zinc Finger protein genes (OsSCZF1 and OsSCZF2), involved in drought, salt and cold stress responses in rice. The expression of OsSCZF1 was induced by drought and NaCl treatments, whereas OsSCZF2 was induced by drought and cold. GFP localization analyses showed that OsSCZF1 seemed to move between the nucleus and cytoplasm, while OsSCZF2 was mainly localized in the nucleus. Transgenic plants over-expressing OsSCZF1 showed a lesion mimic phenotype upon maturity, had an increased expression of pathogenesis related genes and were sensitive to salicylic acid and insensitive to jasmonic acid. Transgenic plants over-expressing OsSCZF2 were short in height, exhibited reduced seed setting and improved tolerance to drought and cold stress. A number of biotic and abiotic stress related genes were up-regulated in OsSCZF2 over-expressing rice plants. These results demonstrate that OsSCZF1 and OsSCZF2 encode functional proteins involved in modulating stress tolerance in rice.

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Reversible protein phosphorylation plays a role in regulating various signal transduction pathways, and is achieved by the combined activities of kinases and phosphatases. Plant PP2Cs constitute a large and diverse family of protein phosphatases compared with other eukaryote PP2Cs. Among them, group A PP2Cs are one of the best characterized PP2Cs, in which they act as negative regulators in ABA signaling pathways. However, little is known about enzymatic and signaling role of the other groups. Here, we characterized a novel isoform of Arabidopsis PP2C (designated AtPP2CF1), which belongs to group E. AtPP2CF1 was expressed in vascular tissues, guard cells and root tips, and this protein was localized in cytoplasm and nucleus. The physiological significance of AtPP2CF1 will be discussed.

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Glutamate is now well established as important signaling molecule in mammalian central nervous system. However, for a long period, it is believed that this signaling pathway does not function in plant. In past decade, glutamate receptors were found on root and mediated Ca flux through cell membrane. In previous meeting, we showed that glutamate induced stomatal closure. At this meeting, we will report result on agonist and knock-down strain of glutamate receptor.
D-serine is well known as agonist of animal glutamate receptor, but it still remains unknown whether it functions in plant glutamate receptors. Stomatal assay showed that D-serine function synergistically in glutamate-induced stomatal closure, suggesting that it plays as agonist of glutamate receptor. Several T-DNA knock-down strains of glutamate receptor were used. The knock-down of AtGLR1 and 3 showed same phenotype on glutamate-induced stomatal closure. However, in the AtGLR2.6 knock-down strain, ABA induced stomatal closure, but glutamate did not , suggesting that this receptor operate in guard cell.

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Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological process. Despite the wealth of information gathered over the last decade, the mechanisms of NO action are poorly known. We now assess the possibility that nitrated cGMP (8-nitro-cGMP) functions in guard cell signaling. At previous meeting, we reported that nitrated cGMP induce stomatal closure. At this meeting, to assess the possibility that nitration of cGMP occur in guard cells, immunocytochemical analyses were performed using a monoclonal antibody against 8-nitro-cGMP. We detected strong fluorescence in guard cells treated with ABA and NO donor. Nitric oxide synthase inhibitor diminished ABA-induced immunofluorescence and NO-scavenger diminished the immunofluorescence induced by ABA and NO. Furthermore, guanylate cyclase inhibitor also cause decrease of ABA- and NO-induced immunofluorescence. These results indicate that 8-nitro-cGMP is formed in an NO- and cGMP-dependent manner.

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